Method to identify tumor suppressor genes

ABSTRACT

This invention provides a method of identifying a tumor suppressor gene of a cell(s). Analogous methods to identify tumor suppressors in normal cells and to identify genes associated with unknown genetic defects are also described. The feasibility of the title method was demonstrated by observing the effects of caffeine acid phenethyl ester on oncogene-tranformed CREF cells. In a second series of expts., human papillomavirus 18-transformed CREF cells were transfected with human fibroblast cDNA cloned into a pMAM-neo vector which allows dexamethasone-inducible expression. After growth in the presence of G418, neomycin resistant transformed CREF cells were obsd. Application of dexamethasone resulted in appearance of cells with normal phenotype.

This application is a 371 of PCT/US95/07738 filed Jun. 15, 1995 which isa continuation-in-part of U.S. application Ser. No. 08/260,326, filedJun. 15, 1994 now abandoned, the contents of which are herebyincorporated by reference.

The invention disclosed herein was made with Government support underNCI/NIH Grant No. CA35675 from the Department of Health and HumanServices. Accordingly, the U.S. Government has certain rights in thisinvention.

Throughout this application, various references are referred to bynumber within parentheses. Disclosures of these publications in theirentireties are hereby incorporated by reference into this application tomore fully describe the state of the art to which this inventionpertains. Full bibliographic citation for these references may be foundat the end of this application, preceding the claims.

BACKGROUND OF THE INVENTION

The carcinogenic process is complex and often involves changes in theexpression of two contrasting genetic elements, i.e., positive actingoncogenes and negative acting anti-oncogenes (tumor suppressor genes)(for reviews see references 1-3). Compounds displaying selectivetoxicity toward transformed cells overexpressing different classes ofoncogenes could prove useful as potential antitumor agents and asreagents for identifying cellular targets susceptible to modification bytransforming oncogenes.

Cancer is often a consequence of changes in the expression of a numberof genes. These include, dominant-acting oncogenes, tumor suppressorgenes, genes affecting cell cycle and genes affecting genomic stability.In the case of tumor suppressor genes, the ability to identify andisolate these elements have proven difficult often involving extensivegene mapping and technically complex and many times unsuccessfulmolecular approaches. Prior to the art described in this invention, nosimple and efficient way of identifying and cloning tumor suppressorgenes has been available. The currently described approach is simple andeffective in directly identifying potentially novel human tumorsuppressor genes and directly cloning these genes. The approach, termedinducible suppression cDNA cloning, is useful in identifying bothoncogene specific suppressor genes and global oncogene-independent tumorsuppressor genes.

Current knowledge of tumor suppressor genes indicate that they oftenfunction as negative regulators of cell growth. Inherent in thisoperational definition of a tumor suppressor gene is the obviousimplication that expression of a tumor suppressor gene in a target cellmay evoke a loss of proliferative ability. This possibility has beendemonstrated directly by reintroducing cloned tumor suppressor genesthrough DNA-transfection into tumor cells, i.e., growth and oncogenicityare suppressed. The growth inhibitory effect of tumor suppressor geneshas prevented the previous development of functional assays permittingisolation of cells expressing novel tumor suppressor genes(anti-oncogenes).

SUMMARY OF THE INVENTION

This invention provides a method of identifying a tumor suppressor geneof a cell(s) which comprises the following steps: a) obtaining cDNA froma normal cell(s); b) preparing a library from the cDNA of step (a),wherein the cDNA is under the control of an inducible expression controlsystem which also carries a selectable gene; c) introducing the vectorlibrary into a population of cell(s) expressing a transformed phenotype;d) placing the introduced transformed cell(s) from step (c) inconditions permitting expression of the cDNA and an effectiveconcentration of an appropriate selection agent to select the cell(s)expressing the selectable gene; e) identifying the cell(s) which expressthe normal phenotype; and f) analyzing the cell(s) so identified so asto characterize the DNA and thus identify the tumor suppressor gene.

Analogous methods to identify tumor suppressors in normal cells and toidentify genes associated with unknown genetic defects are alsodescribed.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C and 1D illustrate the morphology of CREF (A), Ha-ras-transformed CREF (B) and Ha-ras plus Krev-1-transformed CREF clones HKB1 (C) and HK B2 (D). Monolayer cultures were fixed in formaldehyde andstained with Giemsa at approximately×120.

FIGS. 2A, 2B, 2C, and 2D, illustrate the morphology of H5hr1-transformedCREF (A2) (A), a human fibroblast cDNA-induced morphological revertantH5hr1-transformed A2 CREF clone (A2/Hu-Rev/cl 5) (B), v-src-transformedCREF (v-src/cl 1) (C) and a flat v-src revertant CREF clone(v-src/A2-Hu-Rev/cl 3) (D). Monolayer cultures were fixed informaldehyde and stained with Giemsa at approximately×120.

FIG. 3 illustrates the anchorage-independent growth of CREF and CREFcells transformed by diverse oncogenes and transformation-suppressorgenes. Agar cloning efficiency (mean±S.D.) for triplicate samplesinoculated at different cell densities was determined as previouslydescribed (15). Replicate studies of agar growth varied by ≦15%.

FIGS. 4A, 4B, and 4C illustrate the northern analysis of steady-statemRNA in CREF and viral oncogene-transformed CREF cells. A 20-μg aliquotof total cellular RNA was run on a 1.0% agarose gel and transferred to anylon filter. Blots were hybridized with the indicated multiprime³²p-labeled gene probe. Filters were stripped and rehybridized with amultiprime ³²P-labeled GAPDH probe. (A) Expression of v-raf and GAPDHmRNA in CREF and v-raf-transformed CREF (v-raf/cl IIb). (B) Expressionof v-src and GAPDH mRNA in CREF, v-src-transformed CREF (v-src/cl 1) andflat revertant v-src-transformed CREF (v-src/A2 Hu-Rev/cl 3). (C)Expression of A5 E1A, A5 E1B and GAPDH mRNA in CREF, H5hr1-transformedCREF (A2) and flat revertant H5hr1-transformed CREF (A2/Hu-Rev/cl 5).

FIGS. 5A and 5B illustrate the expression of the HPV-18 and HPV-51 inCREF and HPV-18- and HPV-51-transformed CREF cells by RT-PCR. (A)Expression of HPV-18 in CREF, HPV-18-transformed CREF (HPV-18/cl T2) andHPV-51-transformed CREF (HPV-51/cl Al). (B) Expression of HPV-51 inCREF, HPV-18-transformed CREF (HPV-18/cl T2) and HPV-51-transformed CREF(HPV-51/cl A1). The specific E6 primers used for detecting HPV-18 andHPV-51 mRNA and the description of the RT-PCR procedure may be found inMaterials and methods.

FIGS. 6A, 6B, 6C and 6D illustrate the effect of CAPE on the growth of(A) CREF, (B) v-raf-transformed CREF, (C) HPV-18-transformed and (D)HPV-51-transformed CREF. Cells were seeded at 2×10³/3.5-cm plate, andapproximately 16 hours later, the medium was changed and 0, 0.5, 1, 3,5, 10 or 20 μg/ml CAPE added. Cell numbers from triplicate plates weredetermined at days 1, 2, 4, 6, 8, 10, 12 and 14. The medium wasexchanged and the appropriate concentration of CAPE added every 4-5days. Results are the average for triplicate plates which varied by≦10%.

FIGS. 7A, 7B, 7C and 7D illustrate the effect of CAPE on the growth of(A) Ha-ras- transformed CREF (Ha-ras), (B) Ha-ras plusKrev-1-transformed CREF (HK B1), (C) Ha-ras plus Krev-1-transformed nudemouse tumor-derived CREF (HK B1-T) and (D) Ha-ras plusKrev-1-transformed nude mouse lung metastasis-derived CREF (HK B1-M).Experimental details are as described in the legend to FIG. 6A-6D. Celldescriptions may be found in Materials and methods.

FIGS. 8A, 8B, 8C and 8D illustrate the effect of CAPE onH5hr1-transformed CREF (A2), a human fibroblast cDNA-inducedH5hr1-transformed revertant A2 CREF clone (A2/Hu-Rev/cl 5),v-src-transformed CREF (v-src/cl 1) and a v-src-transformed flatrevertant CREF clone (v-src/A2-Hu-Rev/cl 3). Experimental details are asdescribed in the legend to FIG. 6. Cell descriptions may be found inMaterials and methods.

FIG. 9 illustrates the PCR amplification of the unique Krev-1 generegion from CREF, Ha-ras-transformed CREF, Ha-ras plusKrev-1-transformed CREF, Ha-ras plus Krev-1 nude mouse tumor-derived andHa-ras plus Krev-1 metastasis-derived cells. Experimental details can befound in Materials and methods. Lane designations are as follows: M, DNAsize-marker; 1, Ha-ras plus Krev-1 metastasis-derived clone, HK A3-M; 2,Ha-ras plus Krev-1 transformed clone, HK A3; 3, Ha-ras plus Krev-1metastasis-derived clone, HK B2-M; 4, Ha-ras plus Krev-1 nude mousetumor-derived clone, HK B2-T; 5, Ha-ras plus Krev-1 transformed clone,HK B2; 6, Ha-ras plus Krev-1 metastasis-derived clone, HK B1-M; 7,Ha-ras plus Krev-1 nude mouse tumor-derived clone, HK B1-T; 8, Ha-rasplus Krev-1 transformed clone, HK B1; 9, Ha-ras-transformed CREF; and10, CREF.

FIG. 10 illustrates DNA filter hybridization analysis of v-src in CREF,v-src-transformed CREF (v-src/cl 1) and v-src-transformed flat revertantCREF (v-src/A2-Hu-Rev/cl 3), Cellular DNA was cleaved with therestriction endonuclease PstI, 40 μg of DNA was electrophoresed on a 1%agarose gel, transferred to a nylon filter and hybridized with amultiprime ³²P-labeled v-src probe.

FIG. 11 illustrates a scheme for the inducible suppression of cDNAcloning.

FIG. 12 illustrates a modified IscClon strategy.

FIG. 13 illustrates a modified IscClon strategy (antisense approach).

FIG. 14 illustrates a modified lscClon strategy (tetracycline responsivepromoter).

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method of identifying a tumor suppressor geneof a cell(s) which comprises the following steps: a) obtaining cDNA ormRNA from a normal cell(s); b) preparing cDNA from the cell(s) if mRNAis obtained in step (a); c) preparing a library from the said cDNA,wherein the cDNA is under the control of an inducible expression controlsystem which also carries a selectable gene; d) introducing the vectorlibrary into a population of cell(s) expressing a transformed phenotype;e) placing the introduced transformed cell(s) from step (d) inconditions permitting expression of the cDNA and an effectiveconcentration of an appropriate selection agent to select the cell(s)expressing the selectable gene; f) identifying the cell(s) which expressthe normal phenotype; and g) analyzing the cell(s) so identified so asto characterize the DNA and thus identify the tumor suppressor gene.

In this invention, cDNA from normal cells may be obtained directly fromthe cells. First, targeted cells may be infected with retroviral vectorswhich carry appropriate primer. cDNA may now be synthesized within thecell. The synthesized cDNA can therefore be obtained directly from thecell.

In an embodiment of this invention, the inducible expression controlsystem comprises an inducible promoter. In another embodiment, theinducible expression control system comprises a repressible promoter.

An alternative method of achieving controlled expression of genesinvolves the use of a modified tetracycline-repressible, bacterialtetracycline operator/repressor promoter system originally described byGossen, M. and Bujard, H. (“Tight control of gene expression inmammalian cells by tetracycline-responsive promoters”, Proc. Natl. Acad.Sci. USA, 89:5547-5551, 1992). This system uses two plasmid, pUHD15-1and pUHD10-3. pUHD15-1 expresses a chimeric protein containing atetracycline repressor fused to the activation domain of the herpesvirus transcriptional activator, VP-16. The hybrid protein allowstransactivation (tTA) of minimal promoters fused to the tetracyclineoperator sequences (tetO). Plasmid pUHD10-3 has a synthetic promoterwith tandem repeats of a tetracycline operator (tetO) and a CMV-minimalpromoter. The promoter in this plasmid is the target for activation bythe tTA transcriptional activator. In the presence of tetracycline (1μg/ml), this promoter is silent because tetracycline inhibits theability of the tTA transactivator protein to bind to the tetO sequences.Elimination of tetracycline from the culture medium results intranscription of the gene controlled by this promoter in pUHD10-3. Inthis manner, cells containing the appropriate constructs can beexperimentally manipulated to either express or not express normal humancDNAs by removal or addition of tetracycline. These constructscontaining normal human cDNAs in an antisense orientation can be used toidentify suppressor genes that when inhibited result in acquisition of atransformed phenotype by normal human cells. This approach can beapplied directly to normal human cDNA libraries (either 3′-random primercDNA or poly(dT) cDNA) (see below), cloned in a sense and antisenseorientation, for use as part of the inducible suppression cDNA cloning(IScClon) strategy for identifying growth controlling and transformationsuppressing tumor suppressor genes.

In one preferred embodiment of the method described above the cellsexpressing an transformed phenotype are CREF or CREF Trans 6 and may betransformed by either an adenovirus type 5 or the E1A region of theadenovirus type 5. In addition the cells expressing a transformedphenotype may also be transformed by an adenovirus or a retrovirus.

The transformed cells in step (c) of the method described above may betransformed by at least one oncogene or multiple oncogenes. The oncogenemay be H-ras, K-ras, N-ras, v-src, v-raf, HPV-18 or HPV-51. The oncogenemay be a membrane oncogene such as erb B1, erb B2, erb B3, c-fms, hst,kit, c-sis/PDGF-B and trk. Cytoplasmic oncogenes include c-ab1, bcr,c-fes/fps, fyn, raf, ras and src. Other suitable oncogenes includenuclear oncogenes such as PRAD-1, erb-A, c-fos, c-jun, jun B, jum-D,mdm2-, c-myb, c-myc, B-myc, L-myc and N-myc.

A further embodiment of this invention is a method of identifying atumor suppressor gene of a cell(s) which comprises the following steps:a) obtaining cDNA or mRNA from a normal cell(s); b) preparing cDNA fromthe cell(s) if mRNA is obtained in step (a); c) preparing an antisenselibrary from the said cDNA, wherein the cDNA is under the control of aninducible expression control system which also carries a selectablegene; d) introducing the antisense library into a population of normalcell (s); e) placing the transfected normal cell(s) from step (d) inconditions permitting expression of the antisense cDNA and an effectiveconcentration of an appropriate selection agent to select the cell(s)expressing the selectable gene; f) identifying the cell(s) which expressthe transformed phenotype; and g) analyzing the transformed cell(s) soidentified so as to characterize the antisense cDNA and thus identify.the corresponding tumor suppressor gene.

In the methods described above the cell(s) identified in step (f) may beisolated and cultured under conditions so as to isolate and characterizethe DNA and thus identify the tumor suppressor gene.

The invention provides for a method of identifying a gene in a cell(s)associated with an unknown genetic defect having a characteristicphenotype, which comprises the following steps: a) obtaining cDNA ormRNA from a normal cell(s); b) preparing cDNA from the cell if mRNA isobtained in step (a); c) preparing a library from the said cDNA, whereinthe cDNA is under the control of an inducible expression control systemwhich also carries a selectable gene; d) introducing the library into apopulation of cell(s) containing the unknown genetic defect having acharacteristic phenotype; e) placing the cell(s) from step (d) inconditions permitting expression of the cDNA and an effectiveconcentration of an appropriate selection agent to select the cell(s)expressing the selectable gene; f) identifying the cell(s) which expressthe a normal phenotype; and g) analyzing the cell(s) so identified so asto characterize the DNA and thus identify the gene associated with theunknown genetic defect. The cell(s) identified in step (f) may beisolated and cultured under conditions so as to isolate and characterizethe DNA and thus identify the gene associated with the unknown geneticdefect.

The gene in the cell(s) having an unknown genetic defect may be acell(s) from a human tumor cell line or from primary human tumorisolates.

The unknown genetic defect may be associated with the following cancers,oral, esophagus, stomach, colon, rectum, liver, pancreas, larynx, lung,melanoma, skin, breast, cervix uteri, uterus, ovary, prostate, bladder,kidney, brain, non-hodgkin's lymphoma, hodbkin's disease, multiplemyeloma and leukemia.

In one embodiment of the invention the inducer is removed from thecell(s) identified and isolated in step (e) prior to culturing thecell(s) in the methods described above. The inducible promoter may beZn²⁺ metallothionein promoter, metallothionein-1 promoter, humanmetallothionein IIA promoter, lac promoter, laco promoter, mouse mammarytumor virus early promoter, mouse mammary tumor virus LTR promoter,triose dehydrogenase promoter, herpes simplex virus thymidine kinasepromoter, simian virus 40 early promoter or retroviralmyeloproliferative sarcoma virus promoter.

In one preferred embodiment the inducible promoter is a mouse mammarytumor early virus promoter. The promoter may be contained in a plasmid,an adenoviral vector or a retroviral vector.

The selectable gene may be neomycin phosphotransferase, hygromycin,puromycin, G418 resistance, histidinol dehydrogenase or dihydrofolatereductase gene. One preferred embodiment of the invention is aselectable gene which is a neomycin phosphotransferase gene.

The tumor suppressor gene(s) identified by the methods described abovemay be operatively linked to a promoter of RNA transcription. Furtherembodiments of the invention include: a vector which comprises the tumorsuppressor gene; a virus comprising the tumor suppressor gene; apolypeptide encoded by the tumor suppressor gene and an antibody capableof binding to the polypeptide.

Further embodiments of the invention include a non-human mammal whosegerm cells or somatic cells contain a recombinant tumor suppressor geneintroduced into the mammal at an embryonic stage. In additions theinvention provides for a method of treating cells ex vivo whichcomprises contacting cells with the vector comprising the tumorsuppressor gene so as to transform the cells and express the tumorsuppressor gene discovered by the methods herein (see Leder, P. et al.,U.S. Pat. No. 5,175,383, Krimpenfort, P. J. A. et al., U.S. Pat. No.5,175,384, Wagner T. E. et al., U.S. Pat. No. 5,175,385 and U.S. Pat.No. 4,736,866).

As used herein the term identify or characterize the gene i.e. the tumorsuppressor gene includes such methods of identification by fluorescenceis situ hybridization, PCR, other nucleic acid probes and isolating,amplifying and sequencing such genes. Such methods are well known tothose skilled in the art.

As used herein the term a gene associated with a unknown genetic defectmeans and includes a gene which causes a genetic defect. It includesalso genes which are indicative or characteristic of a genetic defect.

The active component of the popular folk medicine propolis, caffeineacid phenethyl ester (CAPE) (5), displays increased toxicity towardcloned rat embryo fibroblast (CREF) cells transformed by adenovirus type5 (Ad5) or the A5 E1A transforming gene versus untransformed CREF cells(5, 6). Employing CREF cells transformed by a cold-sensitive A5 E1A geneand an Ad5 E1A gene under the transcriptional control of a mouse mammarytumor virus promoter, evidence has been presented indicating that CAPEtoxicity is a direct consequence of expression of the E1A-inducedtransformed phenotype (6). Transformation of the established rat embryocell line, Rat 6, with the Ha-ras oncogene was also shown to increasethe sensitivity of these cells to CAPE (6). CAPE and several additionalcaffeine acid esters inhibit azoxymethane-induced colonic preneoplasticlesions and ornithine decarboxylase, tyrosine protein kinase andlipoxygenase activities associated with colon carcinogenesis (7-9). Inaddition, CAPE exerts a dose-dependent growth suppressive effect onhuman colon adenocarcinoma, melanoma and glioblastoma multiforme cells(7, 10). In the human melanoma system, growth suppression was associatedwith the acquisition of morphological changes and the induction of cellsurface antigenic changes suggesting a more differentiated phenotype(10). In contrast, at doses inducing growth suppression and cytoxicityin A5 E1A-transformed CREF or human tumor cells. CAPE was ineffective inaltering the proliferative ability of normal human skin fibroblasts (6).

The mechanism by which CAPE induces its selective toxicity towardoncogene-transformed rodent cells and human tumor cells is not presentlyknown. Further investigation of the phenomenon of CAPE-induced growthsuppression and toxicity in oncogene-transformed rodent cells was ofinterest. For this invention CREF cells transformed by different classesof oncogenes have been employed. Evidence is presented indicating adirect relationship between CAPE sensitivity and transformation inducedby diverse-acting oncogenes, including Ha-ras, HPV-18, HPV-51, v-raf andv-src. By using the Krev-1 tumor suppressor gene, which is 50%homologous to Ki-ras and blocks the transforming activity of Ha-ras andKi-ras transformed cells at a post-transcriptional level (11-13), adirect relationship between expression of the transformed state, asopposed to the presence of the p21 Ha-ras oncogene-encoded protein, andCAPE sensitivity is also demonstrated. Additional studies have focusedon human expression vector cDNA-library-induced revertant H5hr1- andv-src-transformed CREF cells that also display increased resistance toCAPE-induced toxicity versus their transformed counterparts. Takentogether this indicate that the ability of CAPE to induce growthsuppression and toxicity in transformed cells is a direct consequence ofexpression of the transformed phenotype as opposed to simply thepresence of oncogene-encoded transforming proteins.

The technique of inducible suppression cDNA cloning eliminates thesepitfalls and results in the isolation of tumor suppressor genes capableof suppressing the transformed and oncogenic phenotype of oncogenetransformed cells. This approach that is outlined on FIG. 11 is based onthe use of an inducible promoter to selectively regulate expression of atumor suppressor gene. In the example shown, the promoter is a mousemammary tumor virus long terminal repeat sequence that is responsive todexamethasone (DEX). The same approach can be used in conjunction withpromoters responsive to other agents, i.e., Zn²⁺-induciblemetallotheionein promoter, IPTG-inducible (Lacswitch, Stratagene)promoter, etc. By growing cells in DEX, transcription of the human cDNAstably integrated into the target cell genome is induced, whereasexpression is extinguished when DEX is removed from the medium. Thisallows the identification (+DEX) (revertant flat morphology) andisolation (−DEX) (wild-type transformed morphology) of cells containingpotential human tumor suppressor genes. By using target cells containingsingle or multiple oncogenes, or by multiple passage of the same cDNAtumor suppressor gene through cells containing different activatedoncogenes, the currently described approach results in theidentification of tumor suppressor genes with the capacity to revertspecific oncogenic transforming events and/or tumor suppressor genesthat can induce a global suppression of transformation (i.e., reversionof the transformed phenotype in cells containing different activatedoncogenes, multiple activated oncogenes or undefined gene-induced orepigenic transformation related changes).

The current protocol should be very effective in isolating novel classesof tumor suppressor genes. Once identified, the novel tumor suppressorgenes will prove valuable for numerous purposes. Including, designinggene-based strategies for reversing the oncogenic phenotype (adenoviral-or retroviral-based vectors carrying gene replacement constructs);identifying the proteins encoded by the novel tumor suppressor genes(enabling the development of potentially useful therapeutic reagents anddiagnostic monoclonal antibodies); tumor screening and gene localizationstudies (diagnostic applications); and therapeutic intervention based onthe development of rationally designed drugs capable of blockingspecific biochemical pathways defective in cells displaying alteredsuppressor gene functions. By defining the precise genes and the encodedproducts involved in tumor suppression, it will also be possible toidentify potentially novel and critical pathways mediating theneoplastic process. With this information in hand, it will be feasibleto design more effective therapeutic modalities to treat cancer and todevelop approaches (and reagents) to directly reverse the consequencesof oncogene activation and tumor progression.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS First Series of Experiments Materials and Methods

Caffeine Acid Phenethyl Ester

CAPE was synthesized as described by Grunberger et al. (5) and waskindly provided by Drs. T. Doyle and H. Wong, Bristol-Myers Squibb Co.,Wallingford, Conn.

Cell Culture Systems

The CREF cell line is a clonal derivative of the F2408 Fischer ratembryo fibroblast cell line (14, 15). Ha-ras transformed CREF cells(Ha-ras) were obtained following transfection of CREF cells with theHa-ras (T24) oncogene and isolating a focus of cells displaying atransformed morphology (16, 17). Ha-ras/Krev-1 cells, containing theHa-ras and Krev-1 gene, were obtained by cotransfecting Ha-ras cellswith a hygromycin resistance gene (pRSV1.1) (18) and selecting cellsresistant to hygromycin and displaying a reversion in morphology to thatof untransformed CREF cells (19). The Ha-ras/Krev-1 clone HK B1 was used(19). Additionally, an HK B1 nude mouse tumor-derived clone (HK B1-M)and a lung metastasis derived clone (HK B1-M) were analyzed for CAPEsensitivity (19). HPV-18- and HPV-51-transformed CREF cells wereobtained following transfection with the cloned E6/E7 region of HPV-18(20) and HPV-51 (21), respectively, and isolating morphologicallytransformed foci. CREF cells transformed by v-raf and v-src wereobtained by transfecting CREF cells with the appropriate viral oncogeneand isolating morphologically transformed foci (22, 23). A2 is acold-sensitive host-range mutant, H5hr1, transformed CREF clone (24). A2cells are tumorigenic in both nude mice and syngeneic rats (25, 26).Morphological revertant of A2 cells, such as A2/Hu-Rev/cl 5, wereobtained following transfection with a human expression vector libraryconstructed in the PMAM-neo vector. The cloned cDNA inducing reversionin morphology of A2 cells was isolated and transfected intov-src-transformed CREF in morphologically revertant v-src cells, such asv-src/A2-Hu-Rev/cl 3. All cell lines were grown in Dulbecco's modifiedEagle's medium containing 5% fetal bovine serum (DMEM-5) at 37° C. in a5% CO₂-95% air-humidified incubator.

Growth in Monolayer Culture and Anchorage-Independent Growth Assays

For monolayer growth, CREF and the various oncogene-transformed CREFcells were seeded at 2×10³/3.5 cm plate, and approximately 16 hourslater, the medium was exchanged and 0, 0.5, 1, 3, 5, 10 or 20 μg/ml CAPEadded. Cell numbers from triplicate plates were determined at days 1, 2,4, 6, 8 10, 12 and 14. The medium was exchanged and the appropriateconcentration of CAPE added every 4-5 days. For agar cloning studies,cells were seeded at 1×10³ and 5×10⁴/6-cm plate in 0.4% Noble agar on a0.8% Noble agar base layer, both containing DMEM-5. Cultures were refedevery 4 days with 0.4% Noble agar containing DMEM-5. Colonies >0.1 mm indiameter were identified with a calibrated grid under an Olympusinverted phase-contrast microscope after 21 days.

PCR Analysis

To demonstrate the presence and retention of increased copies of theKrev-1 gene in the HK B1, HK B1-T and HK B1-M cell lines PCR analysiswas employed. Cellular DNA was isolated and 40 μg was cleaved with therestriction enzyme BamHI. DNA samples were electrophoresed on a 1%agarose gel overnight and the approximate 1.8 kb fragment was isolatedfrom the gel. This DNA fragment was extracted with phenol:ethanol andprecipitated in ethanol. A total of 5 μg of DNA was used as the PCRtemplate. The primers employed to identify the unique Krev-1 regions(from nucleotide 556 and nucleotide 840) were:5′TATTCTATTACAGCTCAGTCCACG3′ (Seq. ID No. 1) and5′AGGCTTCTTCTTTTCCACTGGTGT3′ (Seq. ID No. 2). DNA samples were PCRamplified (34 cycles: 1 minute, 94° C., 1 minute, 60° C., 1 minute, 70°C.) after addition of the appropriate Krev-1 region primers. Thepredicted 285 nt fragment was detected after electrophoresis in a 1%agarose gel and ethidium bromide staining.

DNA and RNA Analysis

High-molecular-weight DNA was isolated from the transformed CREF celllines as described (15). The presence of viral DNA sequences in theseDNA samples was determined by DNA filter hybridization analysis asdescribed (15, 24). Total cytoplasmic RNA was isolated from cells usingthe guanidinium-thiocyanate/cesium chloride method as described byChirgwin et al. (27) Steady-state levels of the Ads E1A, Ads E1B, v-rafand v-src mRNAs were determined by Northern blot analysis oftotal-cytoplasmic RNA hybridized with appropriate random-primed³²P-labeled probes (28). Northern blots were also probed with a³²P-labeled GAPDH gene (29) to verify similar mRNA expression in thevarious cell types. Presence of HPV-18 and HPV-51 E6 gene expression inappropriate transformed CREF cells was determined by reversetranscription-polymerase chain reaction (RT-PCR) as described byAbdollahi et al. (30). Total cytoplasmic RNA was treated with 0.5 unitsDNase (Boehringer-Mannheim Biochemicals)/μg RNA in 15% glycerol—10 mMTris, pH 7.5—2.5 mM MgCl₂—0.1 mM EDTA—80 mM KCl—1 mM CaCl₂ and 1 unit/mlRNasin (Promega®) at 30° C. for 10 minutes. RNA was extracted withphenol-chloroform, precipitated with sodium acetate/ethanol and RNApellets were resuspended in diethylpyrocarbonate-treated H₂O. One μg oftotal RNA was reverse transcribed with 200 units of murine leukemiavirus reverse transcriptase (Bethesda Research Laboratories) in 20 μlcontaining 1 mM deoxyribonucleotide triphosphates—4 mM MgCl₂—10 mM Tris,pH 8.3—50 mM KCl—0.001% gelatin, and 0.2 μg oligo-dT primer. Sampleswere diluted to 100 μl with buffer containing 0.2 mM deoxyribonucleotidetriphosphates. 2 MM MgCl₂10 mM Tris, pH 8.3, 50 mM KCl and 0.001%gelatin. Fifty pmol of each primer, 1.5 units Taq DNA polymerase(Perkin-Elmer Cetus) were added and samples were covered with mineraloil, heated at 95° C. for 5 minutes and subjected to 20 cycles of PCR ina Perkin-Elmer Thermal Cycler using 2 minutes denaturation at 95° C., 1minute annealing at 55° C. and 4 minutes polymerization at 72° C. Afterextraction with chloroform, 20 Ag of products were electrophoresed,blotted onto nylon filters and hybridized with an HPV-18 E6- or HPV-51E6-specific probe. The template primers for HPV-18 E6 were5′CTGCGTCGTTGGAGTCTTTCC3′ (Seq. ID No. 3) and 5′TTTGAGGATCCAACACGGCGA3′(Seq. ID No. 4) (20) and the template primers for HPV-51 were5′GGGAATTCCTTCACAGTCCATCGCCGTTG3′ (Seq. ID No. 5) and5′GGGGGATCCAACACCATGTTCGAAGACAAG3′ (Seq. ID No. 6) (21).

CAPE Induces Growth Suppression/Toxicitv in CREF Cells Transformed byDiverse-Acting Oncogenes

CREF cells transformed by wild-type and mutant adenovirus type 5 (Ad5)are sensitive to CAPE-induced growth suppression and toxicity (6). Todetermine if this effect is unique to Ad5 -induced transformation ofCREF cells or represents a more general phenomenon associated with thetransformed phenotype, CREF cells were transformed by a series of viraloncogenes, including v-raf, HPV-18, HPV-51, Ha-ras and v-src.Transformation by the various viral oncogenes resulted in morphologicaltransformation (FIGS. 1A-1D and 2A-2D) and acquisition ofanchorage-independence (FIG. 3).

To ensure that CREF cells transformed by the different viral oncogenesexpress appropriate genetic information, Northern blotting wasperformed. CREF cells transformed by v-raf or v-src contained theappropriate viral mRNA (FIG. 4A and 4B). Similarly, previous studieshave indicated that CREF cells transformed by Ha-ras (CREF-ras), CREFcells transformed by Ha-ras and cotransfected with Krev-1 (HK B1) andtumor-derived HK-B1 (HK B1-T) and metastasis-derived HK-B1 (HK B1-M)cells produce Ha-ras mRNA and elevated levels of the rasoncogene-encoded product, p21 (19). In addition, the H5hr1-transformedCREF clone, A2, reverted to a more contact-inhibited morphology (FIGS.2A-2D) following a transfection with a human fibroblast expressionvector cDNA library (A2/Hu-Rev/cl 5) continued to produce both A5 E1Aand E1B mRNAs (FIG. 4C). Expression of the transformation related E6mRNA in HPV-18- and HPV-51-transformed CREF cells was demonstrated byRT-PCR (FIGS. 5A and 5B). These observations indicate that the variouscell lines used were transformed by the specific viral oncogene used andthey express appropriate. viral oncogene-encoded genetic information.

The effect of CAPE on the growth of CREF and CREF cells transformed byv-raf, HPV-18 and HPV-51 is shown in FIGS. 6A-6D. In contrast to CREFcells, which display increases in cell number even when exposed to 15μg/ml of CAPE, 10 μg/ml of CAPE is cytostatic toward v-raf/cl IIb andHPV-18/cl T2cells and 15 μg/ml of CAPE is cytostatic toward HPV-51/cl A1cells. Twenty μg/ml of CAPE is cytotoxic toward HPV-18/cl T2andHPV-51/cl A1 cells. CREF cells transformed by Ha-ras or v-src are evenmore sensitive to the cytostatic and cytotoxic effects of CAPE. A doseof 3 μg/ml of CAPE is cytostatic toward Ha-ras and v-src cells, whereas5 μg/ml or higher doses of CAPE are cytotoxic toward Ha-ras- andv-src-transformed CREF cells (FIGS. 7A-7D and FIG. 8). Similar patternsof CAPE sensitivity have also been observed using additionalindependently derived Ha-ras-and v-src-transformed CREF clones. Theseresults indicate that CREF cells transformed by diverse acting oncogenesbecome sensitive to CAPE-induced growth suppression and cytoxicity. Adirect correlation between the degree of CAPE sensitivity and expressionof the transformed phenotype is also indicated, i.e., transformed cellsdisplaying enhanced growth in agar are more sensitive to CAPE than cellsdisplaying a lower efficiency of agar growth.

CAPE-induced Growth Suppression/Toxicity Correlates Directly withExpression of the Transformed Phenotype in Ha-ras-transformed CREF Cells

The studies described above indicate that acquisition of the transformedphenotype by CREF cells, irrespective of the transforming viral oncogeneused, results in an increase in sensitivity to CAPE-induced growthsuppression. To determine if reversion of the transformed phenotyperesults in a change in sensitivity to CAPE, Ha-ras and Ha-ras/Krev-lexpressing CREF cells (19) have been used (FIGS. 1B, 1C, 7A and 7B).CREF cells transformed by Ha-ras, Ha-ras/cl 5, are morphologicallytransformed, grow with approximately a 38% efficiency in agar and theyinduce both tumors and metastases in nude mice and syngeneic rats (FIGS.1 and 3) (19). In contrast, CREF cells transformed by both Ha-ras andKrev-1 display a reversion in morphology to a more normal CREF-lifephenotype, a reduction in anchorage independence and a suppression intumorigenic and metastatic potential (19) (FIGS. 1 and 3). HK B1 cellsdo, however, induce both tumors (HK B1-T) and lung metastases (HK B1-M)in nude mice after a long latency period (19). Unlike HK BE parentalcells, which display a similar pattern of gene expression asuntransformed CREF cells, HK B1-T and specifically HK B1-M cells displaya reversion in their gene expression to that of Ha-ras cells (19). HKB1-T and HK B1-M cells retain the original Krev-1 gene as indicated byPCR analysis (FIG. 9), continue to synthesize Krev-1 mRNA (19) andHa-ras mRNA (19) and like HK B1 cells continue to synthesize theHa-ras-encoded p21 protein (19). These results indicate that the Krev-1gene can modify expression of the transformed state in Ha-ras cells at apost-transcriptional level. In this respect, this model is ideal fordetermining if CAPE-induced changes are related simply to the presenceof the oncogene-encoded products or to the actual status of expressionof the transformed phenotype.

As shown in FIG. 7A, Ha-ras/cl 5cells are sensitive to both CAPE-inducedgrowth suppression and cytotoxicity. In contrast, HK B1 cells, whichdisplay a CREF-like phenotype, acquire resistance to CAPE. In fact, eventhe highest dose of CAPE tested, 20 μg/ml, does not result in a loss ofproliferative ability in HK B1 cells. A similar reversion to CAPEresistance is observed in two additional independent Ha-ras/Krev-1clones, HK B2 and HK A3 (19). Escape from transformation-suppressionfollowing tumor- and metastasis-induction in nude mice results in HKB1-derived cells that have reacquired sensitivity to CAPE-induced growthsuppression and cytotoxicity (FIG. 7B). HK B1-T and HK B1-M cellsexhibit an increase in anchorage independence in comparison with HK B1parental cells (FIG. 3) (19). HK B1-T cells also display an increase inanchorage-independence and they are more sensitive to CAPE than HK B1-Mcells (FIGS. 3 and 7D). These results indicate that sensitivity to CAPEcorrelates directly with expression of the transformed state in CREFcells, as opposed to the mere presence of oncogene-encoded geneproducts.

CAPE-induced Growth Suppression/Toxicity Correlates Directly withExpression of the Transformed Phenotype in H5hr1- and v-src-transformedCREF Cells

The studies described above using CREF cells transformed by Ha-ras andHa-ras plus Krev-1 indicate a direct correlation between the transformedphenotype and CAPE sensitivity. For further investigation of thisrelationship, two transformation-revertant systems have been used. Usinga modification of the strategy described by Kitayama et al. (11) whichhas resulted in the identification and isolation of the Krev-1suppressor gene, an H5hr1-transformed CREF clone, A2, was transfectedwith an expression vector library containing cDNAs from normal humanskin fibroblasts cloned into the pMAMneo vector. Cells were thenselected in medium containing G418 and 10⁻⁷ M dexamethasone. A series ofG418-resistant colonies, displaying a flat CREF-like morphology, wasisolated and expanded for analysis. The A2 revertant clone, A2/Hu-Rev/cl5were used. This revertant clone displays a CREF-like morphology, has anextended population doubling-time, grows with reduced efficiency in agarand has an increased latency time for tumor formation in nude mice(FIGS. 2 and 3). In contrast to these altered biological properties,both A2 and A2/Hu-Rev/cl 5cells express similar levels of Ad5 E1A andE1B MRNA (FIG. 4C). A2 cells are extremely sensitive to CAPE with 1.5μg/ml resulting in growth suppression and 3 μg/ml or higher levels ofCAPE inducing a cytotoxic effect (6) (FIG. 8). In contrast, although 5,10 and 20 μg/ml of CAPE was cytostatic toward A2/Hu-Rev/cl 5 cells, nocytotoxic effect was apparent in the revertant cells.

The cDNA inducing reversion of the transformed phenotype in A2 cells wasisolated and transfected into v-src-transformed CREF cells. Using asimilar protocol as used in A2 transfections, a series of G418-resistantv-src-transformed CREF colonies displaying a reverted CREF-likemorphology were isolated and expanded for further analysis (FIG. 2). Thev-src-flat revertant, v-src/A2-Hu-Rev/cl 3 were used. In contrast toA2/Hu-Rev/cl 5cells that continue to express A5 gene products,vsrc/A2-Hu-Rev/cl 3 cells do not synthesize v-src MRNA that isdetectable by Northern blotting (FIG. 4B). Southern blotting indicatesthat the v-src gene is still present in v-src/A2-Hu-Rev/cl 3 cells (FIG.10). As observed with A2/Hu-Rev/cl 5versus A2 cells, v-src/A2-Hu-Rev/cl3 cells display increased resistance to CAPE as compared to parentalv-src cells (FIG. 8). These results provide compelling evidenceindicating a direct correlation between expression of the transformedphenotype and CAPE sensitivity.

The ability of diverse acting cellular and viral oncogenes to inducetransformation in primary and established rodent fibroblast cellsindicates that transformation can proceed by different mechanisms(31-33). Irrespective of the transforming agent, transformed fibroblastcells often express similar cellular and biochemical changes (31, 32).Modifications i cellular phenotype associated with transformation ofrodent fibroblasts often include decreased population doubling times,increased saturation densities, acquisition of anchorage-independenceand tumorigenesis in athymic nude mice (31, 32). Compounds that displayenhanced cytostatic/cytotoxic effects toward transformed versus normalcells represent potentially important agents for cancer therapy (34). Itis demonstrated that CAPE displays a selective antiproliferative effecttoward CREF cells transformed by viral oncogenes that display differentmodes of action, including Ads, v-raf, v-src, Ha-ras, HPV-18 and HPV-51.Evidence is also provided that both expression of the transformedphenotype and the degree of expression of transformation (measured byanchorage-independence) as opposed to simply the presence of thetransforming oncogene product, are the mediators of CAPE sensitivity.

Previous studies have shown the effect of CAPE on growth and DNAsynthesis in CREF cells infected, transfected or stably transformed bywild-type or mutant Ad5-transforming genes (6) has been analyzed indetail. CAPE inhibited, in a dose-dependent manner, both de novo andcarcinogen-enhanced transformation of CREF cells by H5hrl. Whentransfected into CREF cells, a cold-sensitive Ads E1A gene only resultedin an inhibition in colony formation by CAPE when cells were grown at apermissive temperature for expression of the transformed phenotype,i.e., 37° C. CAPE was also most effective in inhibiting DNA synthesis inCREF cells containing either a wild-type Ads E1A gene (at 32 or 37° C.)or a cold-sensitive Ad5 E1A gene inducing a transformed phenotype (at37° C.). A direct-requirement for a functional A5 E1A gene, capable ofeliciting the transformed state, and CAPE sensitivity was demonstratedby using CREF cell stably transformed by a cold-sensitive A5 E1A gene oran A5 E1A gene under the transcriptional control of a mouse mammarytumor virus promoter. To determine the effect of the individualtransforming proteins of the A5 A1A gene on CAPE sensitivity, CREF cellswere stably transformed with cDNAs encoding either the 13S or the 12SE1A mRNA, which produce the 289 and 243 amino acid A5 transformingproteins respectively (6). Using these cell lines, it was demonstratedthat CAPE was more growth suppressive toward cells expressing bothtransforming proteins followed by CREF cells transformed with the 13ScDNA and least effective against cells expressing only the 12S cDNA (6).It is demonstrated that CAPE-induced toxicity is eliminated when theH5hr1-transformed CREF clone, A2, is reverted to a more normal CREF-likephenotype by expression of a transfected human fibroblast cDNA.Revertant A2 clone, such as A2/Hu-Rev/cl 5, continue to express both theA5 E1A and E1B transforming genes. These results provide evidence thatCAPE sensitivity in Ad5 -transformed CREF cells is directly dependent onexpression of the transformed state, as opposed to simply the presenceof A5 E1A-transforming gene in A2/Hu-Rev/cl 5 cells.

The conclusion that CAPE-induced toxicity is a consequence of the extentof expression of the transformed phenotype is further supported by thefollowing. Revertant of v-src transformed CREF cells, containing a humancDNA suppressor gene identified in A2/Hu-Rev/cl 5 cells, display astable reversion in transformation-related properties and reacquire anincreased resistance to CAPE-induced growth suppression and toxicity.The v-src revertant clone, v-src/A2-Hu-Rev/cl 3, no longer expressesv-src mRNA, indicating that the increased resistance to CAPE may bemediated by the absence of the transforming oncogene products. In thecase of Krev-1-induced revertant of Ha-ras-transformed CREF cells,acquisition of a reverted transformation phenotype is not associatedwith changes in the levels of the Ha-ras oncogene products (19). Ha-rascells are sensitive to CAPE-induced growth suppression and toxicity,whereas Krev-1 revertant Ha-ras transformed CREF clones, such as HK B1,are resistant to the cytostatic and cytotoxic effects of CAPE. When HKB1 cells escape transformation suppression, following long latency timesin nude mice, tumors (HK B1-T) and lung metastases (HK B1-M) develop.The tumor- and lung metastasis-derived HK E1 clones coordinately displaytransformation-related properties and CAPE sensitivity. In addition, HKB1-T cells display a greater in vitro expression ofanchorage-independence than the HK B1-M clones, and these cells are moresensitive to CAPE. These studies indicate a direct relationship betweenexpression of the transformed state, with and without retention of theoncogene-encoded genetic information, and sensitivity to CAPE.

The mechanism by which CAPE induces its cytostatic and cytotoxicactivity toward CREF cells transformed by diverse acting oncogenes isnot presently known. In the case of Ad5 and human papillomavirus-induced transformation, common cellular genes may provide targetsfor oncoprotein interactions (35, 36). These include, an interactionbetween the Ad E1A- and HPV E7-encoded gene products and specificcellular proteins, such as the retinoblastoma gene product (p105-RB) andthe p105-RB related proteins cyclin A (p107) and p130 (37).

Similarly, the Ads E1B - and HPV E6-encoded gene products specificallytarget the p53 tumor suppressor protein for inactivation (38). Theseobservations have led to the hypothesis that induction of transformationby DNA viruses such as Ad and HPV may involve a direct inactivation ofcellular gene products that normally function as suppressors of thetransformed and oncogenic phenotype. In contrast, current evidencesuggests that Ha-ras induced transformation is a consequence of theintrinsic guanosine triphosphatase activity of the oncogenic ras-encodedprotein, p21, which functions as a component of the guaninenucleotide-binding protein signal transduction pathway in cells (39,40). Similarly, the v-src gene encodes a membrane-associatedtyrosine-specific kinase that is involved in cell signaling pathways andwhich may also involve small guanine nucleotide-binding proteins astarget molecules (41). The transforming gene of murine sarcoma virus3611, v-raf encodes a cytosolic serine/threonine kinase (22). Thecellular homologue of v-raf, c-raf-1, is involved in regulating earlygene expression changes associated with growth-factor stimulation ofcells and acts downstream of ras (22, 42). Since all of the viraloncogenes described above render CREF cells sensitive to theantiproliferative effects of CAPE, apparently multiple and alternativebiochemical changes that ultimately culminate in expression of thetransformed state determine CAPE susceptibility. Furthermore, asindicated above the relative degree of CAPE-sensitivity is also directlyrelated to expression of the transformed state, i.e., cells displayinggreater anchorage-independence are more sensitive to CAPE-induced growthsuppression and toxicity. Additional studies are necessary to define thecommon down-stream target that is shared by all of these oncogenes andwhich serves as the mediator of CAPE sensitivity. As a first step, itwill be necessary to define the site within a cell in which CAPEinitially interacts, i.e., the cell membrane, internal organelles, suchas the mitochondria or the nucleus, specific enzymes, etc. Preliminarystudies using [³H]-labeled CAPE indicate no differential binding betweenCAPE-sensitive cells, such as A2, versus CAPE-resistant cells, such asCREF. These observations suggest that an intracellular target may proveto be the site of interaction with CAPE.

Elucidation of the biochemical changes that render a cell sensitive toCAPE-induced antiproliferative and cytotoxic activities could result inthe identification of common cellular processes altered during oncogenictransformation. This information would prove beneficial in the rationaldesign of chemotherapeutic agents that display antitumor activity towardcancer cells by exploiting common transformation endpoints as targets.In this context, appropriately designed agents would display selectiveactivity toward neoplastic cells that developed as a consequence of theeffects of diverse-acting oncogenes and/or the inactivation ofdiverse-acting tumor suppressor genes.

Second Series of Experiments

Inducible suppression cDNA cloning (IScClon) is a procedure foridentifying and cloning growth inhibitory tumor suppressor genes. Thisapproach can also be used to define genes controlling cellulardifferentiation and cell growth potential (senescence). IScClon is basedon a hypothesis that constitutive expression of specific tumorsuppressor genes in transformed and tumorigenic cell lines can result ina reversion in phenotype to a ore normal cellular state. In manycontexts, this transformation-reversion may correlate with anirreversible loss in proliferative capacity, thereby, preventing theisolation of cells expressing novel tumor suppressor genes, IScClonpermits the identification of cells containing tumor suppressor genes(based on a reversion in cellular morphology under conditions thatinduce tumor suppressor function) and allows growth of tumor suppressorgene containing cells (based on a return to a transformed state and/orresumption of growth under conditions that prevent tumor suppressorfunction). The IScClon approach should prove amenable to identifying andcloning genes mediating reversion of any transforming event, induced byeither known or unidentified genetic changes.

Recent studies have focused on the application of IScClon for revertingthe transformed phenotype of CREF cells transformed by human papillomavirus type 18 (HPV-18) (J. Lin, Z.-z, Su, D. Grunberger, S. G. Zimmer &P. B. Fiher, “Expression of sensitivity to growth phenotype induced bydiverse acting viral oncogenes mediates sensitivity to growth suppressioinduced by caffeine acid phenethyl ester (CAPE)”, Int. J. Oncology,5:5-15, 1994). CREF HPV-18/cl T2cells were transfected with a humanfibroblast random 3′-primer or poly (dT) cDNA expression library clonedinto a pMAM-neo vector (allowing inducible expression in the expressionin the presence of dexamethasone (DEX)). When grown in the presence ofG418, neomycin resistant-transformed CREF HPV-18/cl T2coloniesdeveloped. Application of DEX for 48 hours permitted the identificationof colonies containing cells with a CREF-life morphology. Removal of DEXand further growth resulted in specific colonies that degenerated,remained morphologically normal or reverted back to the transformedphenotype. Both morphologically normal (constitutive in the absence ofDEX) and inducible reverted (normal in the presence of DEX andtransformed in the absence of DEX) colonies have been isolated and arebeing characterized.

Additional studies have been performed using the cold-sensitivehost-range type 5 adenovirus mutant, H5hr1, transformed CREF clone, A2.A2 cells were transfected with a human fibroblast random 3′-primer orpoly (dT) cDNA expression library cloned into a pMAM-neo vector(allowing inducible expression in the presence of dexamethasone (DEX)).Growth in the presence of G418 results in neomycin resistant-transformedA2 colonies. Addition of DEX for 48 hours permitted the identificationof A2 colonies reverting to a more normal CREF-like morphology. As withHPV-18-transformed CREF cells, removal of DEX and further growthresulted in specific colonies that degenerated, remained morphologicallynormal or reverted back to the transformed phenotype. Bothmorphologically normal A2 (constitutive in the absence of DEX) andinducible reverted A2 (normal in the presence of DEX and transformed inthe absence of DEX) colonies have been isolated and are beingcharacterized.

The studies briefly described above suggest that both DEX-inducible andDEX-constitutive morphological revertant transformed cells will provevaluable in identifying genetic elements with the capacity to revert thetransformed and oncogenic capacity of tumor cells. The modified IScClonstrategy is shown in FIG. 12. The modified IScClon using antisense cDNAsshown in FIG. 13. The modified IScClon using tetracycline responsivepromoters is shown in FIG. 14.

Construction of Random Unidirectional Linker-Primer, 3′ Random PrimercDNA and Poly(dT) cDNA, Libraries from Normal Human Skin FibroblastCells

For the inducible suppression cDNA cloning (IScClon) approach, both3′-random primer and poly(dT) primer cDNA libraries are constructed fromnormal human skin fibroblast mRNA. The procedures are as described byStratagene® and in detail in P. G., Reddy, Z.-z. Su and P. B. Fisher,“Identification and cloning of genes involved in progression oftransformed phenotype”, Gene and Chromosome Analysis, K. W. Adolph (Ed),Methods In Molecular Genetics, Vol. 1, Academic Press, San Diego,Calif., pp 68-102, 1993; and H. Jiang and P. B. Fisher, “Use of asensitive and efficient subtraction hybridization protocol for theidentification of genes differentially regulated during the induction ofdifferentiation in human melanoma cells”, Mol. Cell. Different.,1(3):P285-299, 1993.

Total cellular RNA from normal human skin fibroblasts is isolated by theguanidinium isothiocyanate/CsCl centrifugation procedure and poly (A+)RNA was selected following oligo(dT) cellulose chromatography. A totalof 10 μg of mRNA and 5 μg of random-primer (Stratagene, Cat.

# 901151, Jun. 2, 1994 starts reverse transcription (3′-random primercDNA library construction). Alternatively, a total of 5 μg of MRNA and2.5 μg of a oligo(dT) primer (supplied in the Stratagene, ZAP-cDNAsynthesis kit) starts reverse transcription (poly(dT) primer cDNAlibrary construction). Protocols for constructing cDNA libraries aredescribed by Stratagene and Reddy et al (1993) and Jiang and Fisher(1993).

Cloning of the 3′-Random Primer and Poly(dT) Primer cDNA Libraries intoan Inducible Expression Vector (e.g. pMAMneo Vector

Double-stranded phagemid DNAs (from λ ZAP) are prepared from the3′-random primer and poly(dT) human fibroblast (HF) cDNA libraries usingthe mass excision procedure (Stratagene®) as described by Jiang andFisher (1993). Briefly, 1×10⁷ pfu of phagemids containing HF cDNAlibrary are mixed with 2×10⁸ SOLR strain of Escherichia coli and 2×10⁸pfu of E×Assist helper phage in 10 mM MgSO₄, followed by adsorption at37° C. for 15 minutes. After the addition of 10 ml LB medium, thephagemid/bacteria mixture is incubated with shaking at 37° C. for 2hours, followed by incubation at 70° C. for 15 minutes to heatinactivate the bacteria and the λ ZAP phage particles. Aftercentrifugation at 4000 g for 15 minutes, the supernatant is transferredto a sterile polystyrene tube and stored at 4° C. before use.

To produce double-stranded DNA, 5×10⁷ pfu of the phagemids is combinedwith 1×10⁹ SOLR strain of Escherichia coli, which are nonpermissive forthe growth of helper phage and therefore prevent coinfection by helperphage (Ref. 22 from Jiang and Fisher, 1993), in 10 mM MgSO₄, followed byadsorption at 37° C. for 15 minutes.

The phagemidslbacteria are transferred to 250 ml of LB medium containing50 μg/ml ampicillin and incubated with shaking at 37° C. overnight. Thebacteria are isolated by the alkali-lysis method (Ref. 18 from Jiang &Fisher, 1993) and purified through a QIAGEN-tip 500 column (QIAGEN Inc.,Chatsworth, Calif.).

The purified double-stranded cDNA-containing phagemid is digested withthe restriction endonucleases EcoRI and XhoI. The liberated cDNA insertsare isolated from the vector following digesting by electrophoresis init agarose and electroelution. The purified cDNA inserts are ligatedinto the pMAMneo (Clontech) vector that has been incubated with the samerestriction enzymes (EcoRI and XhoI) (inserts: vector=4:1). The ligatedcomplex is then transfected into XL-1 blue strain of Escherichia coliresulting in the production of a dexamethasone (DEX)-inducible humanfibroblast cDNA library. The production and purification of humanfibroblast cDNA inserts/pMAMneo DNA is by the procedure developed byQIAGEN (QIAGEN plasmid Maxi protocol). The human fibroblast expressioncDNA library (50 μ) in 500 ml LB medium with 50 μg/ml ampicillinovernight with shaking at 37° C. Plasmid DNA is isolated by thealkali-lysis method (Ref 18 from Jiang and Fisher, 1993) and purifiedthrough a QIAGEN-tip 500 column.

Identification of Cells Containing Growth and Tumor Suppressor GenesUsing the Inducible Suppression cDNA Cloning (IScClon) Approach.

The IScClon approach is shown in FIG. 12. Approximately 1×10⁶ targetmammalian cells (CREF or CREF-Trans 6 cells containing transfectedoncogenes (including, but not limited to Ad5, A5 mutant, v-src, HPV-18,Ha-ras) or high molecular weight (HMW) human tumor DNA; or human tumorcells) are transfected with 10 μg of plasmid HF cDNA library/pMAMneo DNA(HF cDNA library cloned in a sense orientation for transfer into CREF,CREF-Trans 6 or human tumor cells) by the calcium phosphate, lipofectinor electroporation technique. Transfected cells are replated 48 hr laterand after 24 hr 300 μg/ml of G418 is added. After colonies form,approximately 7 to 21 days depending on the cell type, 10⁻⁶ M DEX isadded. After 24 to 48 hr, plates are scanned microscopically andcolonies displaying a morphologically reverted phenotype (to a normalmorphology with sense inducible cDNAs) are identified and circled. DEXis then removed, colonies are isolated with metal cloning cylinders andmaintained for further analysis as independent cell strains. These cellstrains containing putative human growth suppressing and tumorsuppressor genes can then be used to clone, sequence and characterizethe potentially novel human tumor growth and transformation relatedtumor suppressor genes. At present, two types of cell strains have beenidentified: (a) cell clones that are reverted to a normal phenotype bytreatment with DEX, but retain a normal cellular phenotype even in theabsence of DEX; and (b) cell clones that display a reversible phenotypein the presence (normal phenotype) and absence (transformed phenotype)of DEX (see FIG. 12).

The IScClon approach using antisense cDNA constructs is shown in FIG.13. Approximately 1×10⁶ target normal human cells (skin fibroblast,epithelial, melanocyte, astrocyte, keratinocyte or other normal humancell type) are transfected with 10 μg of plasmid HF cDNA library/mMAMneoDNA (HF cDNA library cloned in an antisense orientation) by the calciumphosphate, lipofectin or electroporation technique. Transfected cellsare replated 48 hours later and after an additional 24 hours 300 to 500μg/ml of G418 is added. Depending on the normal human cell type usedtransfected cells may be plated on feeder-layers consisting ofirradiated CREF cells to improve colony forming efficiency. Aftercolonies form, approximately 14 to 28 days depending on the cell type,10⁻⁶M DEX is added. After 24 to 48 hours, plates are scannedmicroscopically and colonies displaying a morphologically revertedphenotype (to a transformed state) are identified and circled.Alternatively, colonies displaying growth under conditions limitinggrowth of the normal cell type, i.e., removal of specific growthfactors, are also identified. DEX is then removed, colonies are isolatedwith metal cloning cylinders and maintained for further analysis asindependent cell strains. Changes indicating potentially interestingantisense cDNAs include: changes in cellular morphology and growthproperties (morphological transformation, anchorage-independence,acquisition of tumorigenic potential), ability to grow in the absence ofspecific growth factors (insulin, platelet derived growth factor TPA),loss of lineage-specific differentiation markers (melanin production,enzymatic changes, absence of cell surface antigenic markers) andunlimited growth potential (immortality and the loss of senescence).These cell strains containing putative human growth suppressing, tumorsuppressing, differentiation suppressing and/or senescence suppressinggenes can then be used to clone, sequence and characterize thepotentially novel suppressor gene (see FIG. 12).

The IScClon approach using tetracycline (TET) suppressibletetracycline-responsive promoters is shown in FIG. 14. Approximately1×10⁶ target normal mammalian cells (CREF or CREf-Trans 6 cellscontaining transfected oncogenes (including, but not limited to, Ad5,Ad5 mutant, v-src, HPV-18, Ha-ras) or high molecular weight (HMW) humantumor DNA; or human tumor cells are transfected with 10 μg of plasmidPUHD15-1 and 1 μg of pSV2-neo DNA by the calcium phosphate, lipofectinor electroporation technique. Transfected cells are plated 48 hourslater and after an additional 24 hr 300 to 500 μg/ml of G418 is added.G418 resistant colonies are identified after 7 to 21 days, isolated andcell strains expressing the tetracycline repressor fused to theactivation domain of the herpes virus transcriptional activator, VP-16,are identified. These transformed cells are then transfected with 10 μgof plasmid pUHD10-3 containing the HF cDNA library and 1 μg of pRSV1.1DNA is (containing a hygromycin resistance gene) by the calciumphosphate, lipofectin or electroporation technique. Transfected cells(grown in the presence of 1 μg/ml of tetracycline) are replated 48 hrlater and after an additional 24 hr 100 to 400 μg/ml of hygromycin plus1 μg/ml tetracycline is added. Hygromycin resistant colonies areidentified 7 to 21 days later. Tetracycline is removed and after 48 hr,plates are scanned microscopically and colonies displaying amorphologically reverted normal cellular phenotype are identified andcircled. Tetracycline (1 μg/ml) is then added, colonies are isolatedwith metal cloning cylinders and maintained for further analysis asindependent cell strains. These cell strains containing putative humangrowth suppressing and tumor suppressor genes can then be used to clone,sequence and characterize the potentially novel human tumor growth andtransformation related tumor suppressor gene (see FIG. 14).

References

1. Bishop, J. M., (1987) The molecular genetics of cancer. Science235:305-311.

2. Weinberg, R. A., (1991) Tumor suppressor genes. Science254:1138-1146.

3. Marshall, C. J., (1991) Tumor suppressor genes. Cell 64:313-326.

4. Levine, A. J. (1993) The tumor suppressor genes. Annu. Rev. Biochem.62:623-651.

5. Grunburger, D., Banerjee, R., Eisinger, K., Oltz, E. M., Efros, M.,Estevez, V. and Nakanishi, K., (1988) Preferential cytotoxicity on tumorcells of caffeine acid phenethyl ester isolated from propolis.Experiential 44:230-232.

6. Su, Z-z., Grunberger, D., and Fisher, P. B., (1991) Suppression ofadenovirus type 5 E1A-mediated transformation and expression of thetransformed phenotype by caffeine acid phenethyl ester (CAPE). Mol.Carcinogen. 4:231-242.

7. Rao, V. R., Desai, D., Kaul, B., Amin, S. and Reddy, B. S., (1992)Effect of caffeine acid esters on carcinogen-induced mutagenicity andhuman colon adenocarcinoma cell growth. Chem-Biol. Interacts 84:277-290.

8. Rao, V. R., Desai, D., Simi, B, Kulkarni, N., Amin, S. and Reddy, B.S., (1993) Inhibitory effect of caffeine acid esters onazoxymethane-induced biochemical changes and aberrant crypt fociformation in rat colon. Cancer Res. 53:4182-4188.

9. Frankel, K., Wei, H., Bhimani,R., Zadunaisky, J. A., Ferraro, T.,Huang, M. T., Conney, A. H. and Grunberger, D., (1993) Inhibition oftumor promoter-mediated processes in mouse skin and bovine lens bycaffeine acid phenethyl ester. Cancer Res. 53:1255-1261.

10. Guarini, L., Su, Z-z., Zucker, S., Lin, J., Grunberger, D. andFisher, P. B., (1992) Growth inhibition and modulation of antigenicphenotype in human melanoma and glioblastoma multiforme cells bycaffeine acid phenethyl ester (CAPE). Cell Mol. Biol. 38:513-527.

11. Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y. and Noda, M.,(1989) A ras-related gene with transformation suppressor activity. Cell56:77-84.

12. Noda, M., Kitayama, H., Matsuzaki, T., Sugimoto, Y., Okayama, H.,Bassin, R. H. and Ikawa, Y., (1989) Detection of genes with potentialfor suppressing the transformed phenotype associated with activated rasgenes. Proc. Natl. Acad. Sci., U.S.A. 86:162-166.

13. Zhang, K., Noda, M., Vass, W. C., Papageorge, A. G. and Lowy, D. R.,(1990) Identification of small clusters of divergent amino acids thatmediate the opposing effects of ras and Krev-1. Science 249:162-165.

14. Fisher, P. B., Mufson, R. A., Weinstein, I. B. and Little, J. B.,(1981) Epidermal growth factor, like tumor promoters, enhances viral andradiationinduced cell transformation. Carcinogenesis 2:183-187.

15. Fisher, P. B., Babiss, L. E., Weinstein, I. B. and Ginsberg, H. S.,(1982) Analysis of type 5 adenovirus transformation with a cloned ratembryo cell line (CREF). Proc. Natl. Acad. Sci., U.S.A. 79:3527-3531.

16. Boylon, J. F., Jackson, J., Steiner, M., Shih, T. Y., Duigou, G. J.,Roszman, T., Fisher, P. B. and Zimmer, S. G., (1990) Role of the Ha-ras(ras^(H)) oncogene in mediating progression of the tumor cell phenotype(review). Anticancer Res. 10:717-724.

17. Boylon, J. F., Shih, T. Y., Fisher, P. B. and Zimmer, S. G., (1992)Induction and progression of the transformed phenotype in cloned ratembryo fibroblast cells: studies employing type 5 adenovirus andwild-type and mutant Ha-ras oncogenes. Mol. CarcinoQenesis 5:118-128.

18. Su, Z-z., Zhang, P. and Fisher, P. B., (1990) Enhancement of virusand oncogene-mediated transformation of cloned rat embryo fibroblastcells by 3-aminobenzamide. Mol. Carcinogenesis 3:309-318.

19. Su, Z-z., Austin, V. N., Zimmer, S. G. and Fisher, P. B., (1993)Defining the critical gene expression changes associated with expressionand suppression of the tumorigenic and metastatic phenotype inHa-ras-transformed cloned rat embryo fibroblast cells. Oncogene8:1211-1219.

20. Schneider-Gadicke, A. and Schwarz, E., (1986) Different humancervical carcinoma cell lines show similar transcription patterns ofhuman papillomavirus type 18 early genes. EMBO J. 5:2285-2292.

21. Lungu, O., Crum, C. P. and Silverstein, S., (1991) Biologicproperties and nucleotide sequence analysis of human papillomavirus type51. J. Virol., 65:4216-4225.

22. Rapp, U. R., Goldsborough, M. D., Mark, G. E., Bonner, T. I.,Groffen, J., Reynolds, F. H., Jr., and Stephenson, J. R., (1983)Structure and biological activity of v-raf, a unique oncogene transducedby a retrovirus. Proc. Natl. Acad. Sci., U.S.A., 80:4218-4222.

23. Jove, R. and Hanafusa, H., (1987) Cell transformation by the viralsrc oncogenes. Annu. Rev. Cell Biol. 3:31-56.

24. Babiss, L. E., Ginsberg, H. S. and Fisher, P. B., (1983)Cold-sensitive expression of transformation by a host range mutant oftype 5 adenovirus. Proc. Natl. Acad. Sci. U.S.A., 80:1352-1356.

25. Babiss, L. E., Liaw, W-S., Zimmer, S. G., Godman, G. C., Ginsberg,H. S. and Fisher, P. B. (1986) Mutations in the E1a gene of adenovirustype 5 alter the tumorigenic properties of transformed cloned rat embryofibroblast cells. Proc. Natl. Acad. Sci., U.S.A. 83:2167-2171.

26. Su, Z-z., Leon, J. A., Jiang, H., Austin, V. N., Zimmer, S. G. andFisher, P. B., (1993) Wild-type adenovirus type 5 transforming genesfunction as transdominant suppressors of oncogenesis in mutantadenovirus type 5 transformed rat embryo fibroblast cells. Cancer Res.53:1929-1938.

27. Chirgwin, J. M., Przbyla, A. E., MacDonald, R. J. and Rutter, W. J.,(1979) Isolation of biologically active ribonucleic acid from sourcesenriched in ribonuclease. Biochemistry 18:5294-5299.

28. Babiss, L. E., Young, C. S. H., Fisher, P. B. and Ginsberg, H. S.,(1983) Expression of adenovirus E1A and E1B gene products and theEscherichia coli HGPRT gene in KB cells. J. Virol. 46:454-465.

29. Jiang, H., Su, Z-z., Datta, S., Guarini, L., Waxman, S. and Fisher,P. B., (1992) Fludarabine phosphate selectively inhibits growth andmodifies the antigenic phenotype of human glioblastoma multiforme cellsexpressing a multidrug resistance phenotype. Int. J. Oncol. 1:227-239.

30. Abdollabi, A., Lord, K. A., Hoffman-Liebermann, B. and Liebermann,D. A., (1991) Interferon regulatory factor 1 is a myeloiddifferentiation primary response gene induced by interleukin-6 andleukemia inhibitory factor: role in growth inhibition. Cell GrowthDifferent. 2:401-407.

31. Fisher, P. B., (1984) Enhancement of viral transformation andexpression of the transformed phenotype by tumor promoters. In: TumorPromotion and Cocarcinogenesis In Vitro, Mechanisms of Tumor Promotion.T. J. Slaga (ed)., Florida, CRC Press, pp. 57-123.

32. Bishop, J. M., (1991) Molecular themes in oncogenesis. Cell64:235-248.

33. Liotta, L. A., Steeg, P. S. and Stetler-Stevenson, W. G., (1991)Cancer Metastasis and angiogenesis: an imbalance of positive andnegative regulation. Cell 64:327-336.

34. Fisher, P. B., and Rowley, P. T., (1991) Regulation of growth,differentiation and antigen expression in human tumor cells byrecombinant cytokines: Applications for the differentiation therapy ofhuman cancer. In: The Status of Differentiation Therapy of Cancer. S.Waxman, G. B. Rossi and F. Takaku (eds)., New York, Raven Press, pp.201-213.

35. Vousden, K., (1993) Interactions of human papillomavirustransforming proteins with the products of tumor suppressor genes. FASEBJ. 7:872-879.

36. Moran, B., (1993) Interaction of adenoviral proteins with pRB andp53. FASEB J. 7:880-885.

37. Dyson, N., and Harlow, E., (1992) Adenovirus E1A targets keyregulators of cell proliferation. Cancer Surv. 12:161-195.

38. Levine, A. J., (1990) The p53 protein and its interactions with theoncogene products of the small DNA tumor viruses. Virology, 177:419-426.

39. Lowy, D. R. and Willumsen, B. M., (1993) Function and regulation ofras. Annu. Rev. Biochem. 62:851-891.

40. Noda, M., (1992) Mechanisms of reversion. FASEB J. 7:834-846.

41. Pawson, T. and Gish, G. D., (1992) SH2 and SH3 domains: fromstructure to function. Cell 71:359-362.

42. Kolch, W., Heidecker, G., Lloyd, P. and Rapp, U. R., (1991) Raf-1protein kinase is required for growth of induced NIH/3T3 cells. Nature349:426-428.

6 24 base pairs nucleic acid single linear DNA (genomic) unknown 1TATTCTATTA CAGCTCAGTC CACG 24 24 base pairs nucleic acid single linearDNA (genomic) unknown 2 AGGCTTCTTC TTTTCCACTG GTGT 24 21 base pairsnucleic acid single linear DNA (genomic) unknown 3 CTGCGTCGTT GGAGTCTTTCC 21 20 base pairs nucleic acid single linear DNA (genomic) unknown 4TTGAGGATCC AACACGGCGA 20 29 base pairs nucleic acid single linear DNA(genomic) unknown 5 GGGAATTCCT TCACAGTCCA TCGCCGTTG 29 30 base pairsnucleic acid single linear DNA (genomic) unknown 6 GGGGGATCCA ACACCATGTTCGAAGACAAG 30

What is claimed is:
 1. A method of identifying a tumor suppressor geneof a cell(s) which comprises: a) obtaining cDNA or mRNA from a normalcell(s); b) preparing cDNA from the cell(s) if mRNA is obtained in step(a); c) preparing a library from the said cDNA, wherein the cDNA isunder the control of an inducible expression control system which alsocarries a selectable gene; d) introducing the library into a populationof cell(s) expressing a transformed phenotype; e) placing the introducedtransformed cell(s) from step (d) in conditions permitting expression ofthe cDNA and an effective concentration of an appropriate selectionagent to select the cell(s) expressing the selectable gene; f)identifying the cell(s) which express the normal phenotype; and g)analyzing the cell(s) so identified so as to characterize the DNA andthus identify the tumor suppressor gene.
 2. The method of claim 1,wherein the inducible expression control system comprises an induciblepromoter.
 3. The method of claim 1, wherein the inducible expressioncontrol system comprises a repressible promoter.
 4. The method of cclaim 1, wherein the cell(s) identified in step (f) are isolated andcultured under conditions so as to isolate and characterize the DNA andthus identify the tumor suppressor gene.
 5. The method of claim 1,wherein the cells expressing an transformed phenotype are CREF or CREFTrans
 6. 6. The method of claim 5, wherein the CREF cells or CREF Trans6 cells are transformed by either an adenovirus type 5 or the E1A regionof the adenovirus type
 5. 7. The method of claim 1, wherein the cellsexpressing a transformed phenotype are transformed by an adenovirus or aretrovirus.
 8. The method of claim 4, wherein the inducer is removedfrom the cell(s) identified and isolated in step (f) prior to culturingthe cell(s).
 9. The method of claim 2, wherein the inducible promoter isZn²⁺ metallothionein promoter, metallothionein-1 promoter, humanmetallothionein IIA promoter, lac promoter, lacO promoter, mouse mammarytumor virus early promoter, mouse mammary tumor virus LTR promoter,triose dehydrogenase promoter, herpes simplex virus thymidine kinasepromoter, simian virus 40 early promoter or retroviralmyeloproliferative sarcoma virus promoter.
 10. The method of claim 9,wherein the inducible promoter is a mouse mammary tumor virus earlypromoter.
 11. The method of claim 1, wherein the selectable gene isneomycin phosphotransferase, hygromycin, puromycin, G418 resistance,histidinol dehydrogenase or dihydrofolate reductase gene.
 12. The methodof claim 11, wherein the selectable gene is a neomycin phosphtransferasegene.
 13. The method of claim 1, wherein the transformed cells in step(d) are transformed by at least one oncogene.
 14. The method of claim13, wherein the oncogene is H-ras, K-ras, N-ras, v-src, v-raf, HPV-18 orHPV-51.
 15. The method of claim 1, wherein the transformed cells in step(d) are transformed by multiple oncogenes.
 16. A method of identifying atumor suppressor gene of a cell(s) which comprises: a) obtaining cDNA ormRNA from a normal cell(s); b) preparing cDNA from the cell(s) if mRNAis obtained in step (a) c) preparing an antisense library from the saidcDNA, wherein the cDNA is under the control of an inducible expressioncontrol system which also carries a selectable gene; d) introducing theantisense library into a population of normal cell(s); e) placing thetransfected normal cell(s) from step (d) in condition permittingexpression of the antisense cDNA and an effective concentration of anappropriate selection agent to select the cell(s) expressing theselectable gene; f) identifying the cell(s) which express thetransformed phenotype; and g) analyzing the transformed cell(s) soidentified so as to characterize the antisense cDNA and thus identifythe corresponding tumor suppressor gene.
 17. The method of claim 16,wherein the cell(s) identified in step (f) are cultured under conditionsso as to isolate and characterize the DNA and thus identify the tumorsuppressor gene.
 18. A method of identifying a gene in a cell(s)associated with an unknown genetic defect having a characteristicphenotype, which comprises: a) obtaining cDNA or mRNA from a normalcell(s); b) preparing cDNA from cell(s) if mRNA is obtained in step (a);c) preparing a library from the said cDNA, wherein the cDNA is under thecontrol of an inducible expression control system which also carries aselectable gene; d) introducing the library into a population of cell(s)containing the unknown genetic defect having a characteristic phenotype;e) placing the cell(s) from step (d) in conditions permitting expressionof the cDNA and an effective concentration of an appropriate selectionagent to select the cell(s) expressing the selectable gene; f)identifying the cell(s) which express a normal phenotype; and g)analyzing the cell(s) so identified so as to characterize the DNA andthus identify the gene associated with the unknown genetic defect. 19.The method of claim 18, wherein the cell(s) identified in step (f) areisolated and cultured under conditions so as to isolate and characterizethe DNA and thus identify the gene associated with the unknown geneticdefect.
 20. The method of claim 18, wherein the cell(s) having anunknown genetic defect is a cell(s) from a human tumor cell line. 21.The method of claim 18, wherein the cell(s) having an unknown geneticdefect is a cell(s) from primary human tumor isolates.